This RO1 application currently in its second funding cycle requests 5 years of support as a competitive renewal to investigate the molecular and cell biologic mechanisms of angiogenesis in the context of hereditary hemorrhagic telangiectasia (HHT). The PI has been one of the leaders in this field, having identified defects in the TGFbeta receptors endoglin and ALK1 as causing different forms of dominantly inherited HHT about 5 years ago. In the last several years the PI has built upon these findings by screening large numbers of additional patients with HHT and related phenotypes for endoglin and ALK1 mutations, and by carrying out cell culture and cell biologic studies that shed insight into the molecular physiology of angiogenesis. ALK1 is a type I receptor and lies in the same class as ALK5 which is also expressed on endothelial cells. However the two receptors, each of which must form a heterodimer with a type II receptor, have different downstream effectors. The role of endoglin, a type III receptor, is unclear. The PI hypothesizes that ALK1 and ALK5 heterodimers are used for different and somewhat reciprocal phases of angiogenesis, possibly because of different ligand affinity, and that endoglin must either potentate ALK1-associated signaling or interfere with ALK-5-associated signaling. The current application proposes in Aim 1 to develop in vitro systems for ALK1 or ALK5 signaling by adenoviral transduction of a constitutively activated receptor into cultured endothelial cells. The two cell types will be characterized with regard to their global patterns of gene expression and their behavior in three-dimensional culture systems. In Aim 2, parallel studies will be carried out with endothelial cells treated with different concentrations of TGFbeta. Both sets results will be interpreted in the context of an ongoing histopathologic characterization of ALK1 and endoglin knockout mice. In Aim 3, an attempt will be made to examine the role of endoglin directly by coexpression with ALK1 (actually an ALK1-ALK5 chimera) or ALK5 in a cell line that normally lacks both proteins but in which ALK5 signaling can be measured with an appropriate reporter construct. Finally, Aim 4 will attempt to develop mouse models that better mimic pathophysiology of the human disease by construction of double heterozygotes for ALK1 and ALK5 and/or by construction of additional transgenics or knockouts whose design may become apparent during the course of the work.